Two Proteins Targeted to the Same Lytic Granule Compartment Undergo Very Different Posttranslational Processing

Proc. Nadl. Acad. Sci. USA Vol. 86, pp. 7128-7132, September 1989 Immunology Two proteins targeted to the same lytic granule compartment undergo very different posttranslational processing (cytolysin/Na-benzyloxycarbonyl-L-lysine thiobenzyl estrase/immunoelectron microscopy/N-glycosylation/mannose 6-phosphate) JANIS K. BURKHARDT, SUSAN HESTER, AND YAIR ARGON* Department of Microbiology and Immunology, Duke University Medical Center, Box 3010, Durham NC 27710 Communicated by D. Bernard Amos, June 19, 1989 ABSTRACT The granules of natural killer (NK) cells membranes of target cells after exposure to cytotoxic lym- contain cytolysin and serine proteases, proteins that are ex- phocytes (5). Also packaged in thegranules is afamily ofserine pressed specifically in cytolytic cells and are released in re- proteases (6), often called granzymes (7), whose expression is sponse to target binding. We have used immunofluorescence restricted to CTLs and NK cells (6-8). In rat NK cells, two of and immunoelectron microscopy to localize these proteins with these proteases have been classified as Na-benzyloxycar- respect to the various morphological compartments ofgranules bonyl-L-lysine thiobenzyl esterases based on their substrate in a rat NK cell line. Both cytolysin and the proteases are specificity (9). At least one ofthe two, granzyme A, is secreted limited to the core regions of the dense core granules. While during cytolysis (10), but its exact function is not yet known. these proteins are targeted to the same compartment, they A third component of NK granules is a chondroitin sulfate differ markedly in their posttranslational processing. Cytolysin proteoglycan, which is released during the killing process (11). bears N-linked oligosaccharides that are converted to the The presence ofproteoglycans is typical of secretory granules complex type, while the major trypsin-like protease, granzyme in various cell types (3). A, bears only high-mannose-type oligosaccharides. The glycans Although the lytic granules function as secretory granules, of granzyme A, but not those of cytolysin, are modified with they are also similar in many ways to lysosomes. Like phosphomannose moieties. These results suggest that one pos- lysosomes, the granules are acidic (9), although this is also a sible mechanism for packaging proteins into NK granules is the property of secretory granules (3). In addition, NK granules mannose 6-phosphate-dependent lysosomal targeting system. contain many "classical" lysosomal hydrolases, including However, the absence ofthe mannose 6-phosphate modification acid phosphatase, f3-glucuronidase, and aryl sulfatase (12- from cytolysin suggests the existence of yet another targeting 14). Indeed, histochemical assays ofthese enzymes are often system. used to identify large granular lymphocytes. Interestingly, at least some of the lysosomal enzymes are secreted during cytolysis (14, 15), together with the granule-specific proteins. Lytic granules are specialized organelles found in cytolytic This observation implies that the usual sorting of secretory lymphocytes such as cytotoxic T lymphocytes (CTLs), nat- and lysosomal proteins into separate organelles is incomplete ural killer (NK) cells, and lymphokine-activated killer cells. in cytolytic cells, either because the same organelle mediates The binding ofan appropriate target cell, such as a tumor cell both functions or because the population of lytic granules is or a virally infected cell, triggers the directed secretion ofthe actually composed of two distinct organelles. granule contents (1). There is now considerable evidence that Electron microscopic (EM) studies of NK granules show these granule contents can effect the subsequent lysis of the that they are, in fact, morphologically heterogeneous (16). target cell. Although the granules clearly have a specialized Like the condensing vacuoles seen in other secretory cells, secretory role, they also have properties in common with many NK granules contain a homogeneous electron-dense lysosomes. This dual nature raises interesting questions core. Granules having a dense core surrounded by a thin about the biogenesis of lytic granules and the routing of cortex containing membranous material were classified by proteins to them. Neighbour et al. (16) as type I. A second morphological class Granule exocytosis in NK cells is a process of regulated of granules [type II (16)] is larger, more irregularly shaped, and polarized secretion. The Mg2"-dependent binding of NK and resembles multivesicular bodies. Often, a mixed granule cells to targets establishes an axis of polarity through the morphology is observed, where a type I dense core is killer cell and induces a substantial remodeling of its archi- included within a large type II region. tecture (2). This remodeling involves the reorientation of the The relationship among the three types of granules is not Golgi apparatus, cytoskeletal elements, and lytic granules understood. It is not known if the morphological types repre- toward the bound target. The granules then fuse with the sent a maturation sequence of lytic granules. Moreover, it is plasma membrane domain that contacts the target cell and not clear which granules are actually used in cytolysis. EM release their contents. Both the reorientation and the subse- images have been interpreted (16) to show that both type I and quent exocytosis require Ca2", a typical requirement for type II granules can secrete their contents, but this has not regulated secretion (3). been directly demonstrated. Since no nongranule organelle Packaged in the lytic granules are proteins whose expression has been identified as a lysosome in NK cells, it is quite is specific to cytolytic lymphocytes and which are secreted in possible that one type ofgranule corresponds to the secretory response to target binding. Foremost among them is cytolysin lytic granule, while the other type fulfils lysosomal functions. (4), which is analogous to perforin (5). It is thought to effect To address the relationship between lytic granules and target lysis by a Ca2+-dependent oligomerization into cylin- lysosomes, several granule proteins have been localized at drical amphipathic pore structures (1, 5). These pores are the EM level. Perforin was found in the dense core of type I capable of lysing cells and resemble pores observed in the Abbreviations: CTL, cytotoxic T lymphocyte; EM, electron micros- The publication costs of this article were defrayed in part by page charge copy; endo H, endoglycosidase H; NK, natural killer; SBTI, soybean payment. This article must therefore be hereby marked "advertisement" trypsin inhibitor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7128 Downloaded by guest on September 25, 2021 Immunology: Burkhardt et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7129 granules of human CTLs (17). Recently, serine proteases Analysis ofCarboydrates. Serine proteases labeled with 32Pi were also shown to reside in similar granules ofmurine CTLs were immunoisolated from RNK-16 cells by affinity chro- (18). Sulfated proteoglycans have also been localized to type matography on SBTI agarose (Sigma) essentially as de- I granules, most likely to the dense core (11). In contrast, scribed above. The isolated material yielded a single labeled several hydrolytic enzymes are excluded from the dense core band on NaDodSO4/PAGE, which comigrated with the ma- and are localized in the cortical region of type I as well as in jor antigen of the anti-granzyme A (data not shown). After type II granules (12-14). Taken together, these observations digestion to completion with endo H, the samples were suggest biochemical as well as morphological compartmen- chromatographed on a Bio-Gel Pa (200-400 mesh) gel fil- talization of lytic granules. tration column (120 x 1 cm) in 50 mM ammonium acetate (pH To understand this compartmentalization better, we have 7.0). The column was calibrated with 14C-labeled purified examined the biosynthesis of two cytolytic lymphocyte- oligosaccharides (gift of M. Snider, Case Western Reserve specific proteins, cytolysin and granzyme A. In particular, University) and with nitroiodophenylacetyl. The excluded we asked if they behave like lysosomal proteins or like and included volumes were determined by using 1251-labeled secretory granule proteins. Moreover, we asked if the two bovine serum albumin and [3H]mannose, respectively. The proteins are targeted to the same or different morphological peak fractions were pooled, lyophilized, resuspended in regions of granules in NK cells. water, and subjected to mild acid hydrolysis, catalyzed by Dowex 50 H' resin (24), at 970C for 16 hr. The sample was MATERIALS AND METHODS dried quickly with an Evapomix apparatus (Buchler Instru- ments) and resuspended in water. The resulting monosac- Cell Culture and Labeling. RNK-16 clone CRC- is an in charides were analyzed by thin-layerchromatography. What- vitro-adapted clone derived from a large granular lymphocytic man K6 silicagel plates were loaded with radioactive samples leukemia that arose spontaneously in aging Fisher rats (19) mixed with individual cold monosaccharides and developed (gift of C. Reynolds, National Cancer Institute, Frederick, with one of two solvents. Solvent A (2-propanol/water, 4:1) MD). This clone retains NK characteristics, including mor- enabled separation of mannose 6-phosphate from glucose phology, surface markers, and killing specificity, and does not 6-phosphate and

Load more